1. Field of the Invention
The invention relates to a device and a process for testing a sample liquid, especially by means of the ELISA process. In particular, this invention is concerned with microfluidic systems or devices with structures which have a size from roughly 1 to 1000 mm and/or cavities with a volume from roughly 1 to 1000 ml each. The following statements apply to devices and processes in which capillary, pressure and/or centrifugal forces act and are especially decisive for operation.
2. Description of Related Art
The term “ELISA” is an English language acronym for “enzyme-linked immunosorbent assay.” In respect to this invention, this term should be understood in the sense of a process in which an enzyme is bound to an analyzed substance, especially to a complex of an analyzed substance and an antibody. By means of the enzyme, in a detection reaction, a substrate is modified or converted into a detection substrate, especially a fluorescing substrate or the like. A quantitative determination of the analyzed substance in the sample liquid is possible by recording the detection substrate. In order to enable high precision and a corresponding measurement range, conventionally, a dilution series of the sample liquid is studied in this way.
To date, the ELISA process has usually been carried out manually or automatically, for example, by means of pipetting robots, on an open pipetting plate with, for example, 96 open receiving chambers. The sample liquid to be tested is repeatedly diluted in succession in the receiving chambers in order to achieve different dilution conditions. Then, the sample liquid is pipetted with different dilution ratios into prepared receiving chambers in which the analyzed substance in the sample liquid can be bound to immobilized antibodies. After a relatively long reaction time, repeated flushing with a washing liquid takes place. Then, an enzyme bonded to a detection antibody is added. The detection antibody binds to a complex consisting of an analyzed substance and an immobilized antibody. Then again, different washing steps are necessary. Then, a substrate is added which is modified or converted by the enzyme into a detection substrate. The detection reaction is very time critical. The detection reaction is stopped, for example, by adding acid. The problem is that this cannot take place at the same time in all receiving chambers in which the detection reaction proceeds, and that, for greater volumes, different delays can occur due to diffusion and/or mixing processes. Finally, the detection substrate is determined, for example, optically, especially by fluorescence measurement or the like. The concentration of the analyzed substance in the sample liquid can be determined from the determined values.
The explained process is very complex and fault-susceptible. In particular, inaccuracies add up due to the host of individual steps. Furthermore, preparation of the receiving chambers for immobilization of the antibody is accordingly complex and is likewise associated with the use of large amounts of liquid. Moreover, the reactions often proceed very slowly due to the large amounts of liquid, and accordingly, large diffusion paths, so that the ELISA process in the form which has been conventional to date is very time-consuming.
The article “Design of a Compact Disk-like Microfluidic Platform for Enzyme-Linked Immunosorbent Assay” by Siyi Lai et al., Analytical Chemistry, Vol. 76, no, 7, Apr. 1, 2004, pp. 1832 to 1837, describes a microfluidic system in the form of a so-called compact disk (CD) for individual ELISA process steps. A sample liquid, a washing liquid, a liquid with a detection antibody and a substrate liquid are added to corresponding receiving chambers, which are routed in succession by the correspondingly varied rotation of the CD into a single assigned reaction chamber for the corresponding reaction. Thus, individual steps can be carried out in the microfluidic system. However, the pipetting effort is not significantly reduced, since compared to the conventional ELISA process, only the repeated washing steps were avoided.
In general, a host of microfluidic systems in the form of CDs are known, in which the liquid flows are controlled by rotation of the CD, therefore by centrifugal forces.
International Patent Application Publications WO 03/018198 A1 (U.S. Pat. Nos. 6,653,625; 6,717,136 and others), WO 03/072257 A1 (U.S. Pat. No. 6,764,818) and WO 2004/061414 A2 (U.S. Patent Application Publication 2004/121450) disclose microfluidic devices in which a liquid, especially a sample liquid, can be routed from a receiving chamber into connected chambers and can be divided into defined individual amounts and/or can be mixed and preferably react with another liquid. Similar microfluidic systems are also known from U.S. Pat. Nos. 6,705,519 and 6,719,682, U.S. Patent Application Publication 2004/0203136 A1, and International Patent Application Publications WO 00/78455 A1 (U.S. Pat. No. 6,706,519) and WO 01/87485 A2 (U.S. Patent Application Publications 2003/232403 and 2002/151078).
U.S. Patent Application Publication 2004/0203136 A1 discloses a process and a device for testing and diluting samples and reaction liquids. Several metering channels are connected via a common channel to a first receiving chamber for a sample and can be filled with the sample. Furthermore, a second receiving chamber for a dilution liquid is connected to a common channel, and thus, to metering channels. With correspondingly strong rotation, the dilution liquid is routed via the common channel into the metering channels so that the metered sample amounts are transferred into the following mixing chambers which are finally filled completely by the dilution liquid which flows afterward. This does not allow optimum or versatile dilution.